Understanding the basic mechanisms regulating the normal proliferation and differentiation of blood cells is important for our appreciation of events resulting in neoplastic transformation and for possible therapeutic considerations in the treatment of cancer. In vitro marrow culture systems provide useful models for investigating cellular interactions and possible regulators of hematopoiesis. The goals of the proposed research program are to: (1)\characterize bone marrow adherent (stromal) cell populations responsible for production of hematopoietic growth factors; (2)\explore the mechanism(s) involved in the regulation of myelopoiesis; and (3)\determine which marrow-derived adherent cell populations can provide support function for the development of in vivo hematopoietic foci. The technique of long-term marrow culture will primarily be employed for in vitro studies of murine marrow stromal cells. Stromal cell characterization will be accomplished using established long-term cultures and separative methods such as differential trypsinization, counterflow centrifugal elutriation, and radioresistance as well as with histochemical stains, functional assays, and immunofluorescent stains for endothelial, fibroblast, and macrophage markers. The Sl/Sl[unreadable]d[unreadable] mouse, which has been shown to have a defective microenvironment, will serve as a model for exploring the nature of stromal cell dysfunction. The capacity of characterized stromal cells to produce hematopoietic growth factors will be examined using standard clonagenic stem cell assays with isolated stromal cells serving as feeder layers. Individual stromal cells will be evaluated for factor production employing a reverse hemolytic plaque assay and antiserum against purified colony-stimulating factor (CSF). This technique allows for the identification of individual factor-producing cells within a mixed population. Mechanisms modulating production and availability of CSF will be investigated to determine if feedback inhibition occurs via factor consumption or release of inhibitory substances by differentiated cells. These experiments will be carried out using factor assays with mixtures of stromal cells and differentiated myeloid cells. Putative inhibitors of hematopoiesis such as lactoferrin, cyclic AMP, and prostaglandins will be assessed in factor assays of isolated and characterized factor-producing stromal cells. Lastly, the hematopoietic support capacity of isolated stromal cells will be determined following ectopic transplantation into syngeneic mice. These studies should yield useful insights into the functional role of the bone marrow microenvironment and local hematopoietic regulatory mechanisms. (M)